The long-term objective of this proposal is to elucidate the mechanism(s) by which polar lipids are flip-flopped across biogenic (self-synthesizing) membranes such as the endoplasmic reticulum (ER). This is an important, unresolved question in membrane biology. Discovering the molecular basis of flipping is essential to understanding how the phospholipid bilayer of biomembranes is propagated, and how the topologically complex syntheses of various glycolipids are executed. The latter processes are required for assembling biologically important cell surface molecules such as N-glycosylated and glycosylphosphatidylinositol (GPI)-anchored proteins in eukaryotes, and O-antigen-modified lipopolysaccharide in Gram-negative bacteria. (Glyco)phospholipid flip-flop does not occur at an appreciable rate in protein-free liposomes but occurs rapidly in the ER via a protein-dependent, metabolic energy-independent, bi-directional, facilitated diffusion process. We hypothesize that specific proteins, biogenic membrane flippases, facilitate the transbilayer diffusion of polar lipids in the ER, including the glycerophospholipids and isoprenoid-based glycolipids that are the focus of this proposal. The characteristics of lipid flip-flop in the ER rule out the participation of ABC transporters that have been identified as potential lipid translocators in other membrane settings. No biogenic membrane flippases have been identified that flip glycerophospholipids and isoprenoid-P-sugars, but compelling genetic evidence points to a membrane protein, Rft1p, as a flippase for dolichol-pyrophosphate based glycolipids in the ER-localized pathway of protein , N-glycosylation. We propose 2 specific aims: to characterize and identify a glycerophospholipid flippase from yeast and rat liver ER and to test biochemically the role of yeast Rft1p in flipping dolichol-PP-based glycolipids. These studies will make use of procedures that we have developed for the functional reconstitution and assay of lipid flip-flop in proteoliposomes generated from a detergent-extract of ER. We anticipate that our results will define a new class of membrane proteins for which no clear prototype currently exists and begin to address our eventual goal of obtaining a molecular definition of the mechanism of lipid flip-flop in biogenic membranes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM071041-01A1
Application #
6929555
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Chin, Jean
Project Start
2005-07-01
Project End
2009-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
1
Fiscal Year
2005
Total Cost
$319,200
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Goren, Michael A; Morizumi, Takefumi; Menon, Indu et al. (2014) Constitutive phospholipid scramblase activity of a G protein-coupled receptor. Nat Commun 5:5115
Holthuis, Joost C M; Menon, Anant K (2014) Lipid landscapes and pipelines in membrane homeostasis. Nature 510:48-57
Malvezzi, Mattia; Chalat, Madhavan; Janjusevic, Radmila et al. (2013) Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat Commun 4:2367
Georgiev, Alexander G; Johansen, Jesper; Ramanathan, Vidhya D et al. (2013) Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport. Traffic 14:912-21
Wragg, Rachel T; Snead, David; Dong, Yongming et al. (2013) Synaptic vesicles position complexin to block spontaneous fusion. Neuron 77:323-34
Jelk, Jennifer; Gao, Ningguo; Serricchio, Mauro et al. (2013) Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1. J Biol Chem 288:20616-23
Levine, Tim P; Menon, Anant K (2013) A protein pair with PIPs inside. Structure 21:1070-1
Anjem, Adil; Imlay, James A (2012) Mononuclear iron enzymes are primary targets of hydrogen peroxide stress. J Biol Chem 287:15544-56
Chalat, Madhavan; Menon, Indu; Turan, Zeynep et al. (2012) Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis. J Biol Chem 287:15523-32
Menon, Indu; Huber, Thomas; Sanyal, Sumana et al. (2011) Opsin is a phospholipid flippase. Curr Biol 21:149-53

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